Multiple mutations in the EPSPS and ALS genes of Amaranthus hybridus underlie resistance to glyphosate and ALS inhibitors

Amaranthus hybridus is one of the main weed species in Córdoba, Argentina. Until recently, this weed was effectively controlled with recurrent use of glyphosate. However, a population exhibiting multiple resistance (MR2) to glyphosate and imazamox appeared in a glyphosate resistant (GR) soybean field, with levels of resistance up to 93 and 38-fold higher to glyphosate and imazamox, respectively compared to the susceptible (S) population. In addition to imidazolinones, MR2 plants showed high resistance levels to sulfonylamino-carbonyl (thio) benzoates and moderate resistance to sulfonylureas and triazolopyrimidines. Multiple amino acid substitutions were found in both target genes, acetolactate synthase (ALS) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), responsible for conferring high herbicides resistance levels in this A. hybridus population. In the case of EPSPS, the triple amino acid substitution TAP-IVS was found. In addition, MR2 plants also showed increased EPSPS gene expression compared to susceptible plants. A Ser653Asn substitution was found in the ALS sequence of MR2, explaining the pattern of cross-resistance to the ALS-inhibitor herbicide families found at the ALS enzyme activity level. No other mutations were found in other conserved domains of the ALS gene. This is the first report worldwide of the target site resistance mechanisms to glyphosate and ALS inhibitors in multiple herbicide resistance Amaranthus hybridus.


Materials and methods
Plant material. Mature seeds of an A. hybridus population suspected of having multiple resistance (MR) to imazamox and glyphosate were collected from 25 plants in a soybean field (RR) that had been treated with IMI (imidazolinone) herbicides and glyphosate for 20 years in an area of the campus of the University of Córdoba (Argentina). The seeds of a susceptible population (S) were also collected from 25 plants in 2016 from a nearby garden (300 m between the MR and S plants) in which no herbicide had ever been applied.
Multiple resistance in A. hybridus was corroborated for the MR population. For this purpose, seeds were germinated, and a thousand seedlings were transplanted in plots (2 m × 5 m) in the experimental field of the University of Córdoba (Spain). The herbicides were applied using a Pulverex backpack sprayer (Agrocor SA, Córdoba, Spain) with a T-coupler for the bar equipped with four flat fan nozzles, which were calibrated to deliver 200 L ha -1 at a spray pressure of 200 kPa at a height of 50 cm from the level of the plant. When the plants reached the four-leaf stage, they were treated with imazamox at 40 g ai ha -1 . Two weeks later, the surviving plants were treated with glyphosate at 1080 g ae ha -1 . Three months later, more than 80% of the plants finished their reproductive cycle, and mature seeds with multiple resistance were harvested and used for the next step. Approximately 250 seedlings were transplanted in trays (40 cm × 60 cm × 15 cm) that contained sand/peat in a ratio of 1:1 (v/v), and the growth and application followed the same conditions described above. Approximately 95% of the treated plants finished their reproductive cycle. Three months later, the mature seeds were collected, cleaned and stored at 4 °C for one month, and the new seeds were designated as the MR2 population. In parallel, 250 seedlings of the population susceptible to herbicides were treated with imazamox (20 g ai ha -1 ) and glyphosate (360 g ae ha -1 ) separately as explained above. Twenty-one days after the application of imazamox and glyphosate separately, 100% of the plants died, indicating the sensitivity to both herbicides. Both MR2 and S seeds were used in dose-response, biochemical and molecular studies to characterize the multiple resistance.
Seeds from the S and MR2 populations were germinated in Petri dishes containing filter paper that was moistened with distilled water. The Petri dishes were placed in a growth chamber at 28/18 °C (day/night) with a photoperiod of 16 h, 850 μmol s -1 photosynthetic photon flux and 80% relative humidity. All seedlings were transplanted in pots (one plant per pot) containing sand/peat at a ratio of 1:1 (v/v) and placed in a greenhouse with the same photoperiod.
Dose-response assays. To quantify the level of glyphosate and imazamox resistance, seeds of the susceptible (S) and resistant (MR2) populations were germinated as described previously in the Plant material section. ALS and EPSPS enzyme activity assays. For both enzyme activity assays two steps were made: extraction and enzymatic reaction.
The ALS enzyme activity was measured as previously described Palma-Bautista et al. 31 by mean the estimation of the product acetolactate after its conversion to acetoin, by decarboxylation in the presence of acid.
ALS activity was assayed by adding 0.05 mL of enzyme extract to 0.1 mL of freshly prepared assay buffer [0.08 M potassium phosphate (KH 2 PO 4 ⁄ K 2 HPO 4 ), pH 7.5, 0.15 M sodium pyruvate, 1.5 mM MgCl 2 , 1000 μM FAD], and increased concentrations of technical-grade imazamox (0, 0.001, 0.01, 0.1, 10, 100, 1000, 10,000 µM). After mixture incubation (37 °C for 1 h), the reaction was stopped by addition of 0.05 mL of H 2 SO 4 (3 M). The reaction tubes were then heated (15 min at 60 °C) to facilitate decarboxylation of acetolactate to acetoin. Acetoin was detected as a colored complex (520 nm) formed after the addition of 0.25 mL of creatine (5 g L -1 , freshly prepared in water) and 0.25 mL of α-naphthol (50 g L -1 , freshly prepared in 5 M NaOH) and incubated (60 °C for 15 min). Background was determined using control vials, in which the reaction was stopped before the incubation, and subtracted.
The herbicides concentrations necessaries to reduce the ALS activity by 50% (I 50 ) were estimated as has been referred before. The tested herbicides were byspiribac, florasulam, flucarbazone and tribenuron-methyl using the same concentrations as the imazamox (except for florasulam in which the maximum was 1000 µM). To know the resistance pattern, it was required determine the relationship between the R I 50 and the S I 50 (R I 50 /S I 50 ), as well as the biotypes resistance factor to each herbicide in-vitro assay. Total protein content was measured using the Bradford method 32,33 . Maximum ALS specific activity (nmol of acetoin mg -1 of protein h -1 ) was determined in the absence of herbicide and expressed as percentage in relation to the control.
For the EPSPS enzyme activity assay the methodology described by Salas et al. 34 was followed. Five grams of leaf tissue from each population were finely powdered and transferred to tubes with 100 mL of cold extraction buffer (100 mM MOPS, 5 mM EDTA, 10% glycerol, 50 mM KCl and 0.5 mM benzamidine), 70 μL of β-mercaptoethanol and 1% PVPP. After agitation and subsequent centrifugation, (NH 4 ) 2 SO 4 in proportion 45% (w/v) was added to the supernatant, the mixture was stirred and then centrifuged. The previous step was repeated to precipitate the protein. All pellets were dissolved in 3 mL of extraction buffer and dialyzed in 2 L of dialysis buffer (30 mm, 1000-MWC dialysis tubing at 4ºC on a stir plate) over 12 h in cold chamber.
For the determination of EPSPS activity the EnzCheck phosphate assay Kit (Invitrogen, Carlsbad, CA). The substrates for the EPSPS enzyme reaction were phosphoenolpyruvate (1.02 mM) and shikimate-3-phosphate (0.41 mM), supplied by Sigma-Aldrich (Madrid, Spain). The assay buffer was composed of 1 mM MgCl 2 , 10% glycerol, 100 mM MOPS, 2 mM sodium molybdate and 200 mM NaF. The EPSPS activity from the populations was determined without and with glyphosate (0, 0.1, 1, 10, 100 and 1000 μM)32,33. EPSPS activity was measured for 10 min at 360 nm in a spectrophotometer (DU-640, Beckman Coulter Inc. Fullerton, USA) to determine the amount of inorganic phosphate (μmol) released, measured in μg -1 total soluble protein (TSP) min -1 . The proteins concentration was determined by the Bradford assay 33 .
All experiments were conducted with three technical replication of each population per glyphosate and ALSinhibiting herbicide concentration and repeated three times. EPSPS and ALS enzyme activity were expressed as percentage of enzyme activity in presence of glyphosate or ALS-inhibiting herbicide, respectively, with respect to the control.

EPSPS and ALS copy number and gene expression. EPSPS and ALS gene copy numbers in the A.
hybridus genomic DNA were determined as previously described by Gaines et al. 9 . The DNA was purified using the Qiagen DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Once extracted, the DNA concentration was quantified using NanoDrop™ 1000 spectrophotometers (Thermo Scientific) to ensure that the concentration and purity were sufficient for further assays.
Young leaf tissue from ten individuals of each A. hybridus population was collected. Total RNA was extracted using TRI Reagent® solution (Molecular Research Center, Inc., Cincinnati, OH) according to the manufacturer's instructions. RNA was treated using the RNase-free DNase Set (Qiagen, Valencia, CA). M-MLV reverse transcriptase (Promega, Madison, WI) was used to generate cDNA with 3 μg of total RNA as template and random The EPSPS and ALS primer pairs used for copy number and gene expression assays were previously described by Gaines et al. 9 . Reactions were performed by using a qRT-PCR Bio-Rad CFX Connect thermal cycler and the following amplification profile: 50 °C for 2 min; 95 °C for 10 min; 40 cycles of 95 °C for 15 s and 60 °C for 1 min; and 95 °C for 15 s. PCRs were set up in 20 µl of SYBR Green PCR Master Mix (BIO-RAD). The ALS and Actin genes were used as reference genes to normalize the EPSPS and ALS qRT-PCR results, respectively. The relative expression levels were calculated from the threshold cycle (Ct) values and the primer efficiencies by the Pfaffl method 35 .
To determine EPSPS and ALS gene copy numbers, the ALS and actin genes were examined as reference genes by the Pfaffl method 35 . Triplicate technical replications were used to calculate the mean and standard error of the increase in EPSPS/ALS gene copy number relative to the ALS/Actin copy number.
Standard dilution curves were prepared for each primer pair to confirm the appropriate efficiency of amplification (E = 100 ± 10%).

EPSPS and ALS gene sequencing.
To achieve the EPSPS and ALS gene sequencing, ten plants from the susceptible (S) and the resistant (MR2) population were used.
A 196-bp DNA fragment from the conserved region of the EPSPS gene was amplified by RT-PCR as previously described by García et al. 16 . EPSPS gene sequencing was conducted by the Sanger method at the SCAI ("Central Service for Research Support") of the University of Córdoba.
A pair of primers previously reported by Osuna et al. 36 was used to amplify the A. hybridus ALS gene fragments containing all the known mutations. Sequencing of the purified genomic DNA was performed in the Genomic Unit Investigation Central Service of Badajoz University, Spain.
Statistical analysis. The herbicide concentration that caused 50% dry weight reduction (GR 50 ) and plant mortality (LD 50 ) and the herbicide concentration that caused 50% inhibition of enzyme activity (I 50 ) were calculated by analysis of nonlinear regression using the following logistic equation: where Y is the dry weight, mortality and enzymatic inhibition in relation to the control, d is the coefficient corresponding to the limit of the upper asymptote, c is the limit of the coefficient of the lower asymptote (fixed at 0 for GR 50 and LD 50 ), b is the slope of the curve, g is the herbicide concentration required to inhibit shoot growth by 50%, and x is the herbicide dose 37 . Nonlinear regression analysis was performed in the R program 3.6.2 with the drc package (Statistical Software) 37,38 . The resistance indices (RI = MR2/S) were computed as MR2-to-S ratios.

Results
Dose-response assays. Amaranthus hybridus plants of the population MR2 exhibited resistance to glyphosate and imzamox in dose-response experiments. As shown in Fig. 1, plant survival and dry weight decreased as the dose of glyphosate increased. The susceptible population was eliminated by using lower glyphosate doses than those commonly used by Argentinian farmers (960 g ae ha −1 ). The GR 50 and LD 50 values estimated for the MR2 population were 2222 and 4508 g ae ha −1 glyphosate, respectively. According to the GR 50 parameter, the RI of the MR2 population was 126 times higher than that of the S population (Table 1).
For the imazamox herbicide (field dose 40 g ai ha -1 ), the results obtained were similar to those obtained with glyphosate; as the imazamox dose increased, plant survival and dry weight decreased (Fig. 2), and the GR 50 and LD 50 values obtained for the MR2 populations were 403 and 798, respectively. In this case, the RI of the MR2 population was 39, indicating that the MR2 population was 46-fold more resistant to imazamox than the S population (Table 2).     (Fig. 3A). As shown in Fig. 3B, EPSPS activity was inhibited in both S and MR2 plants as glyphosate concentrations increased, but this EPSPS activity inhibition occurred at different herbicide concentrations (Fig. 3B), namely, 0.5 and 59 µM glyphosate for the S and MR2 populations, respectively, resulting in an RI (R-to-S ratio) of 111 (Table 3). The A. hybridus S and MR2 plants presented similar ALS basal activity profiles (Fig. 4A). Imazamox inhibited ALS activity in both cases being the dose needed to do that higher in MR2 plants (Fig. 4B). These concentrations were 117 and 3171 µM imazamox for the S and MR2 populations, respectively, resulting in an RI (R-to-S ratio) of 27. (Table 4).

EPSPS and ALS copy number and gene expression. No significant differences were observed in the
EPSPS and ALS gene copy numbers between S and MR2 A. hybridus plants (Fig. 5). Nevertheless, EPSPS gene expression was higher in MR2 plants compared to S population, with expression levels up to 4 times higher (Fig. 6). In most cases an increase in the expression levels of a gene is usually correlated with a higher copy number of the gene, but this is not the current case. In MR2 population the high EPSPS expression levels are not correlated with an EPSPS copy number increment, this suggests that EPSPS gene expression is subjected to a regulation mechanism at transcriptional level.

Mutations in the EPSPS and ALS coding sequences. The point mutations found both in EPSPS and
ALS genes corroborated the resistance to glyphosate and imazamox from whole plant experiments. The multiple resistant A. hybridus population showed the EPSPS triple amino acid substitution Thr102Ile, Ala103Val and Pro106Ser which was previously described by our group 16 with another A. hybridus population that was resistant only to glyphosate, data not shown (GenBank accession number MG595171).
Only a nucleotide point mutation (AGC to AAC) was found in the ALS sequence of MR2 when compared with the corresponding sequence in the S population, resulting in a Ser653Asn substitution (Fig. 7). This explains the pattern of cross-resistance to the ALS-inhibitor herbicide families found at the ALS enzyme activity level among these accessions. No other mutations were found in other conserved domains of the ALS gene (data not shown).

Discussion
Multiple resistance to glyphosate and ALS inhibitor, namely imazethapyr, was previously found in an A. hybridus population from Argentina in 2014 2 . Several years later, in 2018, an A. hybridus population with multiple resistance to glyphosate and to the ALS inhibitor chlorimuron-ethyl, was described in Brazil 2 . Nevertheless, the resistance mechanisms were not described. In this study, multiple resistance to glyphosate and ALS inhibitors in the A. hybridus population from Córdoba, Argentina, has been studied and characterized to decipher the TSR mechanism underlying the high resistance levels shown for the first time. The GR 50 value relative to glyphosate shown by this MR2 population (2222 g ae ha −1 glyphosate) was higher than that of the previous resistant population (Glyphosate-resistant) (1395 g ae ha −1 glyphosate), the results for which were previously published 16 .
These two different populations, MR2 and GR2 (glyphosate resistant population which results were previously published in García et al. 16 ) are carriers of the novel triple amino acid substitution in the conserved region of the EPSPS gene. The difference between them lies in the increased EPSPS gene expression shown by the MR2 population, which was up to 4 times higher in the MR2 population than in the S population. This increment in the EPSPS gene expression was not correlated with an increase in the EPSPS gene copy number. Assuming no differences between S and R plants in copy number, changes in transcriptional regulation might explain the EPSPS overexpression found in this particular population, which would be quite novel 16 . The first case of herbicide resistance induced by EPSPS gene copy number occurred in an A. palmeri population in 2010 7 . In this work, the authors found a 100-fold increase in EPSPS gene copy number, resulting in an up to 40-fold increase in EPSPS expression. These mechanisms, that is, EPSPS overexpression due to increased copy number, have also been described in other species of the Amaranthus genus, such as A. tuberculatus 12 and A. spinosus 10 , as well as in multiple weed species 39,40 . Mutations in the conserved region of EPSPS conferring glyphosate resistance have been previously reported in both mono-and dicotyledonous weeds. The single amino acid substitutions at Pro106 confer glyphosate resistance in several weeds, such as Digitaria insularis, Lolium multiflorum, Amarantus spinosus, A. palmeri and Lolium rigidum 9,[41][42][43] . Single substitutions at Gly101 and Thr102 confer high levels of glyphosate resistance but decrease the volume of the glyphosate-PEP binding site, reducing affinity for PEP 44,45 . The artificial double amino acid change in EPSPS (Thr102Ile and Pro106Ser) known for conferring high glyphosate resistance levels in commercial transgenic maize lines, has also been reported in Eleusine indica 15,46 and Bidens pilosa 47    www.nature.com/scientificreports/ reported for the first time in an A. hybridus population from Cordoba, Argentina 16 , and this mechanism was the only mechanism responsible for the high resistance level (GR 50 of 1395 g ae ha −1 ) shown in this case 16 . The higher resistance level developed by the MR2 population (GR 50 of 2222 g ae ha −1 ) must be due to the presence of an alternative second mechanism, as the EPSPS gene expression level was up to four times higher in MR2 plants than in S plants. ALS point mutations have been widely described in numerous weed species, particularly at 8 positions of the ALS gene 19 . Depending on the ALS amino acid position affected and the specific substitution, variable patterns of cross-resistance between ALS inhibitor classes can occur 49 . Amino acid substitutions of Ala122 or Ser653 conferred resistance to IMI herbicides with low-level resistance to SUs 27,50-52 , whereas substitution of Pro197 conferred resistance to SUs 53 but low or no cross-resistance to IMIs. Substitution of Trp574 or Ala205 conferred broad cross-resistance; 50,54 however, substitution of Ala205 conferred much lower levels of resistance than substitution of Trp574.
In MR2 plants, the single amino acid substitution Ser653Asn is responsible for the high resistance level to IMIs. Although the Ser653 substitution has been described to confer resistance only to IMIs, there are some cases in which broad cross-resistance has been observed. Whaley et al. 22,23 found that the Ser653Asn amino acid substitution conferred resistance to IMIs, pyrimidinyl (thio) benzoates (PTBs) and triazolopyrimidines (TPs) in several Amaranthus hybridus biotypes. This is the first time that TSR mechanisms by means of point mutations for both glyphosate and ALS inhibitors are reported in Amaranthus hybridus. Moreover, an additional TSR mechanism to glyphosate has been found, because MR2 population showed increased EPSPS gene expression. In this regard, new management alternatives must be used, both chemical and non-chemical, to control this A. hybridus population from Córdoba, Argentina. Among chemical options, photosystem I and II, protoporphyrinogen oxidase (PPO) and 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors or auxin-mimic herbicides should be considered to ensure the sustainability of the crop production system.